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Heat-resistant alpha-amylase from bacillus geobacillus stearothermophilus and application thereof

A technology of Geobacillus and thermophilic fat, which is applied in the field of biotechnology and food, can solve the problems of high production cost and low yield, and achieve high thermal stability and good thermal stability

Active Publication Date: 2019-11-08
BAIYIN SINO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of commercial application due to low yield and high production cost, cloning the α-amylase gene from the original strain and expressing it in a heterologous host can effectively solve this difficulty

Method used

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  • Heat-resistant alpha-amylase from bacillus geobacillus stearothermophilus and application thereof
  • Heat-resistant alpha-amylase from bacillus geobacillus stearothermophilus and application thereof
  • Heat-resistant alpha-amylase from bacillus geobacillus stearothermophilus and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] Amplification of the α-amylase gene

[0030] 1.1 Strains and their cultivation

[0031] The α-amylase gene of the present invention is cloned from Geobacillus stearothermophilus.

[0032] Geobacillus stearothermophilus (DSMZ456) of the present invention can be purchased directly from the German Culture Collection of Microorganisms, the strain number is DSMZ 456, and its original source is extracted from sugar beet juice in Austria. Therefore, the Geobacillus stearothermophilus described in the present invention can be obtained through commercial means, and can also be obtained through field collection or other means.

[0033] Glycerol bacteria were inoculated into No. 1 medium, cultured at 55°C for 2 days, and the bacteria were collected for genome extraction.

[0034] 1.2 Genome Extraction

[0035] Refer to the instructions of the Generay Bacterial Genomic DNA Rapid Extraction Kit.

[0036] 1.3 Amplification of the α-amylase gene from Geobacillus stearothermophilus...

Embodiment 2

[0044] Construction and expression of Pichia pastoris recombinant expression vector containing α-amylase

[0045] 2.1 Recombinant expression vector pPIC9K-amy GSM build

[0046] Escherichia coli DH5α host bacteria containing pPIC9K were extracted with AxyPrep Plasmid DNA Extraction Kit (refer to the instruction manual for the operation steps), single enzyme digestion was performed with Not I (purchased from Themo Company), the linear vector fragment was recovered by gel cutting, and then the linear vector fragment was recovered with ClonExpress Ultra One The Step Cloning Kit kit (refer to the instruction manual for the operation steps) was connected to the vector and the target gene (obtained in Example 1), transformed into Escherichia coli DH5α (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), PCR verified positive clones and sequenced, The comparison and analysis of the sequencing results on NCBI showed that the obtained α-amylase gene DNA consists of 165...

Embodiment 3

[0055] α-amylase activity assay

[0056] 3.1 Standard curve drawing

[0057] Take a clean test tube, label the test tube, prepare a glucose concentration gradient solution, add 0.2-1.4mL (at 0.2mL intervals) of 1% glucose solution to the test tube, and use the test tube without glucose as a blank control. Three parallel samples were made for each tube. Add ddH to the test tube respectively 2 O to a total volume of 2.0mL, then add 3mL DNS reagent to the test tube, boil for 15min, immediately add 10mL ddH 2 0 and pre-cooling, measure colorimetrically with a spectrophotometer at a wavelength of 540nm, and write down the optical density values ​​of the samples corresponding to each test tube and calculate the average value, then draw the glucose standard curve.

[0058] 3.2 Determination of enzyme activity

[0059] The enzymatic activity of recombinant α-amylase was measured by DNS termination method.

[0060] reaction system:

[0061]

[0062] React in a water bath at 70...

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Abstract

The invention relates to a heat-resistant alpha-amylase from bacillus geobacillus stearothermophilus and site-specific mutagenesis improvement thereof. According to the heat-resistant alpha-amylase, arecombinant plasmid containing amyGSM and a recombinant plasmid containing mutated M1-amyGSM and M2-amyGSM are successfully constructed, and thus various highly-expressed bacterial strains such as escherichia coli, bacillus, saccharomycetes and molds are obtained. An ecombinant host cell obtained is suitable for expression of an alpha-amylase gene. The optimum pH of the alpha-amylase is 6.5, theoptimum temperature is 70 DEG C, the thermal stability at 70 DEG C is good, and the enzyme activity is still about 50% after heat preservation for 8 h; and glutamate at the163th site is mutated to arginine, after removal of isoleucine and glycine at the 215th site and the 216th site, the higher thermal stability is achieved, and the alpha-amylase still remains the 85.6% enzyme activity after heatpreservation for 30 min at 85 DEG C.

Description

technical field [0001] The invention relates to the fields of biotechnology and food, in particular to a heat-resistant alpha-amylase derived from Geobacillus stearothermophilus and application thereof. Background technique [0002] α-amylase (1,4-α-D-glucan hydrolase, EC 3.2.1.1), as an important starch hydrolase, can specifically hydrolyze the internal α-1 of starch, glycogen or polysaccharide, 4 glycosidic bonds, producing products such as short-chain dextrins, oligosaccharides and glucose, which are widely used in industry, such as brewing, food, medicine and textiles (Kandra L. α-Amylases of medical and industrial importance. Journal of Molecular Structure Theochem, 2003, 666:487-498.). It has been reported that α-amylase can be used to produce fructose syrup, as an anti-aging agent to improve the quality of baked goods, and for important applications such as washing industry, paper industry and production of fuel ethanol (Gupta R, Gigras P, Mohapatra H , et al. Micro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2417C12N15/815C12Y302/01001
Inventor 刘校函俞峰丁少明
Owner BAIYIN SINO BIOTECH
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